Chitosan is a biopolymer of natural origin, derived from chitin which is obtainable from crustacean shell, but can also be obtained from other invertebrates and from fungi. Chitosan is prepared by deacetylation of the N-acetyl glucosamine residues of the chitin polymer, typically by hydrolyzing the N-acetyl linkages with concentrated alkali. By definition, chitosan is generally described as a copolymer of D-glucosamine (D) and N-acetyl-D-glucosamine (A), which is insoluble in water at pH above 6.2—the isoelectric point of the free amine group—but dissolves at pH below about 6.2. Typically, 70-100% of the monomeric units in conventional chitosan copolymer are D-glucosamine, which can be described as 70-100% deacetylated chitosan with a degree of deacetylation of 70-100%. When the degree of deacetylation is lower than about 70%, the chitosan polymer displays different solubility properties, increased bioactivity, and generally higher biodegradability.
Chemical and biological properties of chitosan are directly influenced by the degree of deacetylation (DD) and degree of polymerisation (DP), i.e. the chain length of the polymer. In solution at pH below 6.2, and when amine groups of the D-glucosamine residues are protonated, chitosan is a positively charged polymer. Being an amine, chitosan is a weak base and can form salts with acids, such as carboxylic and mineral acids. Most of these salts are water-soluble. In its natural form, chitin is insoluble in water. However, it can be made water-soluble by partial deacetylation through alkali treatment. Partially deacetylated chitin with DD of 35-50% is soluble in water at a wide range of pH. This form of partially deacetylated chitin has been shown to be bioactive with potential applications in various fields such as in biomedicine, pharmaceuticals, cosmetics, etc.
One of the drawbacks of chitosan preparations with more than 60-70% DD is its tendency to precipitate at pH above 6.2. This limits its spectrum of applications where solubility at neutral to high pH is require. In this respect, the partially deacetylated chitin has greater advantages over chitosan with higher DD, since its solubility profile covers a wider range of pH. It inherits most of the physical-chemical properties of chitosan and possesses higher water holding capacity as compared to normal chitosan. resulting in rapid swelling when in contact with water and posses balanced hydrophilic/hydrophobic properties as compared to regular higher DD chitosan. These properties represent an immense potential in various applications in biomedical, pharmaceutical, cosmetic and other related industries.
Biological activity of chitin and chitosan is abundantly documented in the literature, and growing evidences indicate that bioactivity is increased with lower DD. This goes hand in hand with improved solubility properties at physiological pH.
Purification of chitosan commonly involves a dissolution process so as to remove insolubles or impurities from the solution. This is followed by recovery process through precipitation of chitosan from the solution. The recovery of chitosan in a form of precipitate, can then be washed to neutral pH and to remove salt. This recovery is generally not a problem for chitosan with 55% DD and above, since it can be precipitated easily by increase the solution pH to above 6.2. However, adjustment of pH is not effective for partially deacetylated chitin, usually an organic solvent is needed to aid the precipitation process. Patent No. CN1554267 reported the use of ethanol for washing the polymer and more examples on the use of solvents can be found in patents JP10072502, CN1371922, etc. Alternatively, in a less adequate method, the solution is just filtered and dried, whereby salt will be present in the product (JP2022301).
Chitosan has been shown to be biocompatible and biodegradable, making it an attractive choice as an ingredient in biomaterials for bioengineering applications. Biomaterials are generally defined as synthetic materials used to replace part of a living system or to function in intimate contact with living tissue, and chitosan has generally been considered as a suitable inert component in biomaterial formulations or as a matrix for other substances or ingredients. Chitosan has been suggested as a drug delivery carrier as it can immobilize a large amount of bioactive substances through adsorption or by covalently binding such substances through simple chemical reactions.
WO 2004/028578 discloses a composition for bone formation and bone consolidation in bone extension comprising chitosan, tripolyphosphate and bone morphogenic protein (BMP). Further, US 2003/0124172 discloses a method for producing chitosan-based films comprising a biodegradable polymer and BMP for enhancing osteointegration of dental implants or in traumatic situations.
Bioactivity of chitin-derived materials has been indicated, e.g. in EP 1435976 that discloses chitooligomer compositions comprising heterooligomers of N-acetyl-glucosamine and glucosamine, which are biologically active and are suggested as active ingredients in medicaments for treating conditions in connective tissue, in particular arthritis and osteoarthritis.
In an other patent application it is suggested that chitinase like proteins (CLPs) expressed by the genomes of humans and other vertebrates, represent target receptors involved in the bioactivity of these chitooligomers, inducing a signalling response when binding to the chitooligomers. These chitinase like proteins derive from a family of genes expressing the Family 18 Chitinases in most forms of living organisms. The active side of the Family 18 Chitinases is well preserved in the CLPs, with the exception that most of these proteins have lost their catalytic activity through key mutations in the active side. However, in humans, at least two of these proteins maintain their chitinolytic activity i.e. Acetic Mammalian Chitinase (AMCase) and Chitotriosidase.